• 동의신경정신과학회지
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• 제8권1호
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• pp.95-109
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• 1997

In order to prove the experimental effectiveness of ChongsimOndamTang by categotizing COTⅠ, COTIⅡ, COTⅢ and COTⅣ according to the volume of COT extract,the anticonvulsion action, the antipyretic action, the analgesic action,the sedative action, and the activity of GABA transaminase, the actuvity concentration of GABA,the activity of GAD in GABAergic system comparing data with control group and observation data show the results as follows.1. The anticonvulsion effect on the convulsion induced by strychine it was significantly effective in COTⅣ and the time to death after the occurrence of the convulsion it was significantly effective in COTⅢ and COTⅣ, and the time to death after the occurrence the convulsion induced by the electrical shock of ECT unit it was significantly effective in all sample groups.2. The hypothemic effect was significantly effective in COT Ⅲ after 1 hour and 2 hour and was significantly effective COTⅣafter 1 hour and 4 hour, and the antipyretic effect on the febrile induced by endotoxin it was significantly effective in COTⅢ every 3 hour and was significantly effective in COTⅣ after 3 hour and 4 hour.3. The analgesic effect was significantly effective in COTⅢ and COTⅣ by decreasing the number of writhing syndrome.4. The sedative effect was decreased significantly all in COTⅢ and COTⅣ after 60 min, 90 min and 120 min.5. The activity of GABA transaminase was decreased significantly in COTⅡ and COTⅢ. 6. The activity concrntration of GABA was increased signifivantly in COTⅡ and COTⅢ. 7. The activity of GAD was increased significantly in COTⅡ and COTⅢ. The results show that ChongsimOndamTang can be an effective cure in mice on the anticonvulsion,the antipyretic , the analgesic,the sedative and the control of the GABAergic system in brain, and it can be used of the epilepsy and convulsive diseases clinically.

• Molecules and Cells
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• 제25권1호
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• pp.43-49
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• 2008

Cancer Osaka thyroid, also known as Tpl-2 (Cot) is a member of the MAP3K kinase family and plays a key role in the regulation of the immune response to pro-inflammatory stimuli such as lipopolysaccharide (LPS) and tumour necrosis $factor-{\alpha}$ ($TNF-{\alpha}$). A series of Cot constructs with an N-terminal 6xHis tag were transiently expressed in HEK293 cells: $Cot_{130-399}$ (kinase domain), $Cot_{1-388}$ (N-terminal and kinase do-mains), $Cot_{1-413}$, $Cot_{1-438}$ (containing a putative PEST sequence), $Cot_{1-457}$ (containing both PEST and degron sequences) and $Cot_{1-467}$ (full-length protein). These Cot proteins were pulled down using an anti-6xHis antibody and separated by 2D electrophoresis. The gels were silver-stained and 21 proteins were detected that did not appear, or had substantially reduced intensity, in the control sample. Three of these were identified by MS and MS/MS analysis as Hsp90, Hsp70 and Grp78. Hsp90 appeared to bind to the kinase domain of Cot and this interaction was further investigated using co-immuno-precipitation with both overexpressed Cot in HEK293 cells and endogenous Cot in Hela cells.

• Journal of Microbiology and Biotechnology
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• 제29권9호
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• pp.1383-1390
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• 2019

In this study, we expressed cotA laccase from Bacillus subtilis on the surface of B. subtilis spores for efficient decolorization of synthetic dyes. The cotE, cotG, and cotY genes were used as anchoring motifs for efficient spore surface display of cotA laccase. Moreover, a $His_6$ tag was inserted at the C-terminal end of cotA for the immunological detection of the expressed fusion protein. Appropriate expression of the CotE-CotA (74 kDa), CotG-CotA (76 kDa), and CotY-CotA (73 kDa) fusion proteins was confirmed by western blot. We verified the surface expression of each fusion protein on B. subtilis spore by flow cytometry. The decoloration rates of Acid Green 25 (anthraquinone dye) for the recombinant DB104 (pSDJH-EA), DB104 (pSDJH-GA), DB104 (pSDJH-YA), and the control DB104 spores were 48.75%, 16.12%, 21.10%, and 9.96%, respectively. DB104 (pSDJH-EA) showed the highest decolorization of Acid Green 25 and was subsequently tested on other synthetic dyes with different structures. The decolorization rates of the DB104 (pSDJH-EA) spore for Acid Red 18 (azo dye) and indigo carmine (indigo dye) were 18.58% and 43.20%, respectively. The optimum temperature for the decolorization of Acid Green 25 by the DB104 (pSDJH-EA) spore was found to be $50^{\circ}C$. Upon treatment with known laccase inhibitors, including EDTA, SDS, and $NaN_3$, the decolorization rate of Acid Green 25 by the DB104 (pSDJH-EA) spore decreased by 23%, 80%, and 36%, respectively.

• KSBB Journal
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• 제26권3호
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• pp.199-205
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• 2011

Using tetrameric ${\beta}$-galactosidase as a model protein, anchoring motives were screened in Bacillus subtilis spore display system. Eleven spore coat proteins were selected considering their expression levels and the location in the spore coat layer. After chromosomal single-copy homologous integration in the amyE site of Bacillus subtilis chromosome, cotE and cotG were chosen as possible spore surface anchoring motives with their higher whole cell ${\beta}$-galactosidase activity. PAGE and Wester blot of extracted fraction of outer layer of purified spore, which express CotE-LacZ or CotG-LacZ fusion verified the existence of exact size of fusion protein and its location in outer coat layer of purified spore. ${\beta}$-galactosidase activity of spore with CotE-LacZ or CotG-LacZ fusion reached its highest value around 16~20 h of culture time in terms of whole cell and purified spore. After intensive spore purification with lysozyme treatment and renografin treatment, spore of BJH135, which expresses CotE-LacZ, retained only 1~2% of its whole cell ${\beta}$-galactosidase activity. Whereas spore of BJH136, which has cotG-lacZ cassette in the chromosome, retained 10~15% of its whole cell ${\beta}$-galactosidase activity, proving minor perturbation of CotG-LacZ, when incorporated in the spore coat layer of Bacillus subtilis compared to CotE-LacZ. Usage of Bacillus subtilis WB700, of which 7 proteases are knocked-out and thereby resulting in 99.7% decrease in protease activity of the host, did not prevent the proteolytic degradation of spore surface expressed CotG-LacZ fusion protein.

• Journal of Microbiology and Biotechnology
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• 제27권3호
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• pp.507-513
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• 2017

Bacillus subtilis spores can be used for protein display to engineer protein properties. This method overcomes viability and protein-folding concerns associated with traditional protein display methods. Spores remain viable under extreme conditions and the genotype/phenotype connection remains intact. In addition, the natural sporulation process eliminates protein-folding concerns that are coupled to the target protein traveling through cell membranes. Furthermore, ATP-dependent chaperones are present to assist in protein folding. CotA was optimized as a whole-cell biocatalyst immobilized in an inert matrix of the spore. In general, proteins that are immobilized have advantages in biocatalysis. For example, the protein can be easily removed from the reaction and it is more stable. The aim is to improve the pH stability using spore display. The maximum activity of CotA is between pH 4 and 5 for the substrate ABTS (ABTS = diammonium 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate). However, the activity dramatically decreases at pH 4. The activity is not significantly altered at pH 5. A library of approximately 3,000 clones was screened. A E498G variant was identified to have a half-life of inactivation ($t_{1/2}$) at pH 4 that was 24.8 times greater compared with wt-CotA. In a previous investigation, a CotA library was screened for organic solvent resistance and a T480A mutant was found. Consequently, T480A/E498G-CotA was constructed and the $t_{1/2}$ was 62.1 times greater than wt-CotA. Finally, E498G-CotA and T480A/E498G-CotA yielded 3.7- and 5.3-fold more product than did wt-CotA after recycling the biocatalyst seven times over 42 h.

• 동의신경정신과학회지
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• 제18권1호
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• pp.1-14
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• 2007

Objective : The purpose of this study was to investigate the protective effects of Cheongsimondam-tang(COT) on the animal model of depression induced immobilization stress. Method 1) Male rats were used for this experiment. The subject were divided into 4 groups(1. normal 2. saline solution administered during immobilization stress treatment 3. COT of 100mg/kg administered 4. COT of 400mg/kg administered). 2) Immobilization stress was treated for 1 hours on day. During 2 days of immobilization stress treatment, they were executed forced swimming test, passive avoidance test, elevated plus maze test. Corticosterone in blood were measured Results 1) In EPM test, stress group showed significantly increased anxiety, COT groups showed significantly decreased anxiety. 2) In forced swimming test, COT groups did not show significantly decreased immobilization. 3) In passive avoidance test, stress group showed significantly decreased learning execution, COT groups showed significantly increased learning execution. 4) Stress group showed significantly increase in serum level of corticosterone, COT of 400mg/kg group showed significantly decreased serum level of corticosterone. Conclusion : These results suggest that Cheongsimondam-tang(COT) is effective in the treatment of depression.

• 벤처다이제스트
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• 제2호통권127호
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• pp.28-29
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• 2009

매일 아침 정해진 시간에 출근해 일하고, 퇴근하는 따분한 일상. 일상의 대부분을 회사생활속에서 스트레스의 해소는 물론, 직원 간의 끈끈한 정이 있는 작은 일탈은 약이 된다. 평생교육 및 성인자격증 콘텐츠를 인터넷 등을 통해 서비스하는 이러닝 산업의 선두주자, (주)유비온의 레저동아리 'COT'는 일탈을 꿈꾸는 유비온들의 휴식공간이 되어주는 사내동호회다.

• Journal of Microbiology and Biotechnology
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• 제26권7호
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• pp.1267-1277
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• 2016

Currently, enzymatic synthesis of lactulose, a synthetic prebiotic disaccharide, is commonly performed with glycosyl hydrolases. In this work, a new type of lactulose-producing biocatalyst was developed by displaying β-galactosidase from Bacillus stearothermophilus IAM11001 (Bs-β-Gal) on the surface of Bacillus subtilis 168 spores. Localization of β-Gal on the spore surface as a fusion to CotX was verified by western blot analysis, immunofluorescence microscopy, and flow cytometry. The optimum pH and temperature for the resulting spore-displayed β-Gal was 6.0 and 75℃, respectively. Under optimal conditions, it showed maximum activity of 0.42 U/mg spores (dry weight). Moreover, the spore-displayed CotX-β-Gal was employed as a whole cell biocatalyst to produce lactulose, yielding 8.8 g/l from 200 g/l lactose and 100 g/l fructose. Reusability tests showed that the spore-displayed CotX-β-Gal retained around 30.3% of its initial activity after eight successive conversion cycles. These results suggest that the CotX-mediated spore-displayed β-Gal may provide a promising strategy for lactulose production.

• Journal of Microbiology and Biotechnology
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• 제17권4호
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• pp.677-680
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• 2007

To analyze a cotG-based Bacillus subtilis spore display system directly, $GFP_{UV}$ was expressed on the surface of Bacillus subtilis spores. When $GFP_{UV}$ was fused to the C-terminal of the cotG structural gene and expressed, the existence of a $CotG-GFP_{UV}$ fusion protein on the B. subtilis spore was confirmed by flow cytometry confocal microscopic analysis. When the cotG anchoring motif was deleted, no fluorescence emission was observed under flow cytometry and confocal microscopic analysis from the purified spore, confirming the essential role of CotG as an anchoring motif. This $GFP_{UV}$ displaying spore might be used for another signaling application triggered by intracellular or extracellular stimuli.

• Journal of Power Electronics
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• 제17권5호
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• pp.1298-1307
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• 2017

The constant on-time current-mode controlled (COT-CMC) switching dc-dc converter is stable, with no subharmonic oscillation in its current loop when a voltage ripple in its outer voltage loop is ignored. However, when its output capacitance is small or its feedback gain is high, subharmonic oscillation may occur in a COT-CMC buck converter with a proportional-integral (PI) compensator. To investigate the subharmonic instability of COT-CMC buck converters with a PI compensator, an accurate reduced-order asynchronous-switching map model of a COT-CMC buck converter with a PI compensator is established. Based on this, the instability behaviors caused by output capacitance and feedback gain are investigated. Furthermore, an approximate instability condition is obtained and design-oriented stability boundaries in different circuit parameter spaces are yielded. The analysis results show that the instability of COT-CMC buck converters with a PI compensator is mainly affected by the output capacitance, output capacitor equivalent series resistance (ESR), feedback gain, current-sensing gain and constant on-time. The study results of this paper are helpful for the circuit parameter design of COT-CMC switching dc-dc converters. Experimental results are provided to verify the analysis results.